Method of screening substances serving as the active ingredient in preventives or remedies for neurodegenerative diseases

ABSTRACT

A method for screening substances which are useful as effective components of prophylactic or therapeutic drug for neurodegenerative diseases caused by the binding of an aberrant protein and a valosin-containing protein, wherein the aberrant protein and the valosin-containing protein and the candidate substance are made to coexist, and the substance that shows inhibitory action on the binding of the aberrant protein and the valosin-containing protein is identified, is provided.

TECHNICAL FIELD

[0001] The invention of the present application relates to a method forscreening substances that are useful as the effective components inprophylactic or therapeutic drugs for neurodegenerative diseases and ascreening kit that uses such method. More particularly, the inventionrelates to a method for screening substances that inhibit the binding ofknown aberrant proteins that cause neurodegenerative diseases tovalosin-containing protein (VCP), thereby identifying substances thatare useful as the effective components of prophylactic or therapeuticdrugs for neurodegenerative diseases. The invention further relates to ascreening kit that is based on such method.

BACKGROUND ART

[0002] Neurodegenerative diseases such as Parkinson's disease,Alzheimer's disease, Huntington's chorea, and Machado-Joseph disease,occurs at a rate of one person in 10 thousand to 30 thousand for eachdisease. In Japan alone, the existence of more than 100 thousandpatients (one person per 1,200) has been confirmed (Ministry of Welfare;Journal of Health and Welfare Statistics, 1996). Therefore, much efforthas been made on the research of preventative and therapeutic methods(Borlongan, C. V., Koutouzis, T. K., Sanberg, P. R., Neurosci. Biobehav.Rev. 21, 289-93 (1997); Parnetti, L., Senin, U., Mecocci, P., The wayforward. Drugs. 53, 752-68 (1997); Pasinetti, G. M., Neurobiol. Aging.17, 707-16 (1996)). Studies have found that neurodegenerative diseasesshare a common phenotype. That is, it has been confirmed that theaggregation and accumulation of aberrant proteins, which havedistinctive characteristics such as extended glutamine chain, abnormalconformation, resistance to proteases, β-sheet structure, etc., causethe formation of vacuoles and cell death cells (Kakizuka, A., TrendsGenet. 14, 396-402 (1998)).

[0003] However, the causal matter that is involved in the induction ofaggregation and accumulation of such aberrant proteins, which then causethe formation of vacuoles and cell death, is yet to be identified,thereby delaying the research and development of remedies andprophylactics for neurodegenerative diseases.

[0004] The inventors of the present application have, through assiduouswork, succeeded in identifying the factor that induces the phenotypecommon to such neurodegenerative diseases, and revealed that the factoris avalosin-containing protein (VCP)

[0005] VCP, an AAA (ATPase Associated with various cellular Activities)super family protein existing generally in organisms ranging fromeucaryotic cells to humans, has a weak ATPase activity and has beenconsidered to be involved in various cell functions, particularly signaltransduction (Muller, J M., Meyer, H. H., Ruhrberg, C., Stamp, G. W.,Warren, G., Shima, D. T., J. Biol. Chem. , 274, 10154-10162 (1999);Zhang, L., Ashendel, C. L., Becker, G. W., Morre, D. J., J. Cell Biol.,127, 1871-1883 (1994)). However, VCP was not known to be a factor thatinduces neurodegenerative diseases by binding with aberrant proteins.

[0006] On the basis of this new finding, it is expected that byestablishing a method for screening substances that inhibiting thebinding of VPC and the aberrant protein, substances that are commonlyeffective as therapeutic or prophylactic drugs for a specific disease,as well as for a series of neurodegenerative diseases may be obtained.

[0007] The invention of the present application was made in view of theabove-described situation, and its object is to solve the problems ofthe prior art and to provide a method for screening substances useful aseffective components in prophylactic or therapeutic drugs forneurodegenerative diseases.

DISCLOSURE OF INVENTION

[0008] In order to solve the above-described problems, the presentinvention firstly provides a method for screening substances which areuseful as effective components of prophylactic or therapeutic drug forneurodegenerative diseases caused by the binding of an aberrant proteinand a valosin-containing protein, comprising: making the aberrantprotein and the valosin-containing protein and the candidate substancecoexist; and identifying the substance that shows inhibitory action onthe binding of the aberrant protein and the valosin-containing protein.

[0009] Secondly, the present invention provides the above method forscreening substances, wherein the aberrant protein is a protein thatcontains an extended glutamine-chain; thirdly, the present inventionprovides the above method for screening substances, wherein the aberrantprotein contains an abnormal conformation; fourthly, the presentinvention provides the above method for screening wherein the aberrantprotein is a protease-resistant protein; and fifthly, the presentinvention provide the above method for screening a substance, whereinthe aberrant protein contains a β-sheet structure.

[0010] Further, the present invention sixthly provides theabove-described method of screening substances, wherein the aberrantprotein and the valosin-containing protein and the candidate substanceare made to coexist by mixing in a solution; the present inventionseventhly provides the above method for screening a substance, whereinthe aberrant protein and the valosin-containing protein and thecandidate substance are made to coexist in a cultured cell; the presentinvention eighthly provides the method for screening a substance,wherein the aberrant protein and the valosin-containing protein and thecandidate substance are made to coexist by administering each componentto an animal.

[0011] Furthermore, ninthly, the present invention provides the abovemethod for screening substances, wherein the inhibitory action isdetected by immobilizing the non-labeled protein on a carrier;contacting the aberrant protein and the valosin-containing protein inthe presence of the candidate substance; and detecting the signalgenerated by the labeled protein on the immobilized carrier.

[0012] Also, the present invention tenthly provides a kit for screeningsubstances which are useful as effective components of prophylactic ortherapeutic drug for neurodegenerative diseases caused by the binding ofan aberrant protein and a valosin-containing protein, comprising:

[0013] a carrier on which the valosin-containing protein is immobilized;and a labeled aberrant protein reagent.

[0014] Finally, the present invention eleventhly provides a kit forscreening substances which are useful as effective components ofprophylactic or therapeutic drug for neurodegenerative diseases causedby the binding of an aberrant protein and a valosin-containing protein,comprising: a labeled valosin-containing protein reagent; and a carrieron which the aberrant protein is immobilized.

BRIEF DESCRIPTION OF DRAWINGS

[0015]FIG. 1 is a fluorescence micrograph and an optical micrographshowing the localization of VCP protein and the expressed polyglutaminein the PC12 cells (PC12/Q79) which express the Flag tagged-79 repeatpolyglutamine (Q79) by removing tetracycline in the Example of thepresent invention.

[0016] 1: IntrinsicVCPproteininthePC12 cells (a) fluorescencemicrograph; (b) optical micrograph.

[0017] 2: Fluorescence micrograph 48 hours after the removal oftetracycline (a) The intrinsic VCP stained with an anti-VCP antibody(second antibody=FITC-labeled anti-rabbit antibody) in PC12/Q79; (b)Polyglutamine stained with anti-Flag antibody (second antibody=texasred-labeled anti-mouse antibody) and the DAPI-stained nucleus inPC12/Q79; (c) Superposition of the intrinsic VCP stained with ananti-VCP antibody (second antibody=FITC-labeled anti-rabbit antibody)and polyglutamine stained with an anti-Flag antibody (secondantibody=texas red-labeled anti-mouse antibody) in PC12/Q79; (d) Opticalmicrograph of PC12/Q79.

[0018] 3: Fluorescence micrograph 96 hours after the removal oftetracycline (a) The intrinsic VCP stained with an anti-VCP antibody(second antibody=FITC-labeled anti-rabbit antibody) in PC12/Q79; (b)Polyglutamine stained with anti-Flag antibody (second antibody=texasred-labeled anti-mouse antibody) and the DAPI-stained nucleus inPC12/Q79; (c) Superposition of the intrinsic VCP stained with ananti-VCP antibody (second antibody=FITC-labeled anti-rabbit antibody)and polyglutamine stained with an anti-Flag antibody (secondantibody=texas red-labeled anti-mouse antibody) in PC12/Q79; (d) Opticalmicrograph of PC12/Q79.

[0019]FIG. 2 shows the results of autoradiography for confirming whetherthe binding inhibition of MJDQ79 and VCP occurs when a chaperone protein(Hsp70, or HJD-2) coexists with VCP and GST-MJDQ7 in the Example of thepresent invention. (a) Interaction of GST-MJDQ79 and VCP when the amountof Hsp70 or HJD-2 coexisting with GST and GST-MJDQ79 was changed; withinput of VCP, Hsp70 and HJD-2 expressed as 1. (b) A graph expressinglanes 7, 8, 9, 11 and 12 in FIG. 2a (interaction of VCP with GST-MJDQ79)numerically. (c) Interaction of GST-VCP with MJDQ79 when the amount ofHsp70 or HJD-2 coexisting with GST and GST-VCP was changed; with theinput of MJDQ79, Hsp70 and HJD-2 expressed as 1. (d) A graph inexpressing lanes 10, 11 and 12 in FIG. 2c (interaction of GST-VCP withMJDQ79) numerically.

[0020]FIG. 3 shows the results of autoradiography indicating the bindingof a precursor protein of amyloid β-protein and VCP in the Example ofthe present invention.

[0021]FIG. 4 shows a micrograph of the Lewy bodies in DLB stained withanti-VCP antibody in the Example of the present invention. (▾)

[0022]FIG. 5 shows a micrograph of ubiquitin-positive nuclear inclusionbodies stained in the Example of the present invention. (a) Stained withanti-ubiquitin antibody; (b) Stained with anti-VCP antibody.

BEST MODE FOR CARRYING OUT THE INVENTION

[0023] The inventors of the present application have found, throughassiduous work, that neurodegenerative diseases are induced by thebinding of an aberrant protein to a valosin-containing protein (VCP),which causes degeneration and omission of nerve cells, and completed themethod for screening substances useful as effective components inprophylactic or therapeutic drugs for neurodegenerative diseases of thepresent invention.

[0024] In the invention of the present application, the term aberrantprotein means proteins which aggregate and accumulate in the nerve cellsto cause vacuolization and cell death. For example, aberrant proteinsinclude proteins that contain an extended glutamine chain, proteins thatcontain abnormal conformation, proteins that are protease-resistant andproteins with a β-sheet structure.

[0025] The method for screening substances of the present inventioncomprises making an arbitrary aberrant protein and a candidate substanceand VCP coexist, and confirming whether the binding of the aberrantprotein and the valosin-containing protein is inhibited or not, therebyscreening the substance which would become the effective component inprophylactic or therapeutic drugs for neurodegenerative.

[0026] In this screening method, the method by which the aberrantprotein, VCP and candidate substance are made to coexist is notparticularly limited. For example, an aberrant protein and VCP may bemixed in a solution in the presence of a candidate substance, makingthem coexist in solution, or each component may be incubated in yeast orcultured cells (including bacteria) and made to coexist. In addition,VCP may be made to coexist in a transgenic animal (vertebrate, nematode,drosophila, etc.) in which an aberrant protein is expressed, and acandidate substance may be introduced thereto. The condition forcoexistence such as temperature, concentration, pH, time, etc., is notparticularly limited, as long as the condition is sufficient for theaberrant protein to bind to VCP and for the candidate substance toeffectively inhibit the binding. In particular, a method wherein thecandidate substance is introduced by various means into cells in whichan aberrant protein and VCP coexists, is preferably exemplified.

[0027] Also, the method for confirming whether the binding of theaberrant protein and VCP is inhibited or not by a candidate substance isnot limited. For example, a method wherein the aberrant protein islabeled with a tag such as His or Flag or with radioisotopes such as³⁵S, and made to contact with VCP immobilized on a carrier for a certainperiod of time, after which the presence of the labeled signal on thecarrier is confirmed, a method wherein the labeled aberrant protein andVCP is mixed and incubated, after which the solution is passed through acolumn on which a substance that binds to VCP specifically isimmobilized, and the presence of the labeled signal on the column or inthe eluate is confirmed may be applied. Furthermore, a method ofdetermining whether the inhibition of binding occurs by a candidatesubstance, by observing the occurrence of vacuolization in the cytoplasmof cells expressing an aberrant protein (for example, PC12/Q79: Yasuda,S., Inoue, K., Hirabayashi, M., Higashiyama, H., Yamamoto, Y., Fuyuhiro,H., Komure, O., Tanaka, F., Sobue, G., Tsuchiya, K., Hamada, K., Sasaki,H., Takeda, K., Ichijo, H., Kakizuka, A., Genes Cells 4, 743-756(1999)), by the coexistence of VCP and the candidate substance, a methodof determining the usefulness of a candidate substance by observing thepresence of vacuolization, localization of the aberrant protein and VCPin cells, or change of fluorescence resonance energy transfer (FRET), inthe coexistence of VCP labeled with a fluorescent protein such as GFP,YFP, CFP, etc., cells expressing the aberrant protein, and the candidatesubstance may also be exemplified.

[0028] In the method for screening substances of the present invention,experimental techniques generally known in biochemistry, including GSTpull-down assay and yeast 2 hybrid method may be applied. Further,BIACORE by which the degree of binding can be confirmed by measuringsurface plasmon resonance is preferable for the screening of largequantities of samples. However, methods for screening substances of thepresent method are not limited to the above examples, as long as theinhibition of the binding of an aberrant protein with VCP by a candidatesubstance in the coexistence of the aberrant protein, VCP and thecandidate substance can be confirmed.

[0029] As described above, in the invention of the present application,the method for screening a substance useful as the effective componentfor prophylactic or therapeutic drugs for neurodegenerative diseasescomprises confirming the inhibition of the binding of an aberrantprotein with VCP in the coexistence of the aberrant protein, VCP and acandidate substance. The method exemplified above may be conducted underany conditions employing any cells, reagents, concentrations,temperatures, pHs, etc.

[0030] In the method for screening substances of the present invention,a candidate substance that is to be screened may be a naturallyoccurring material such as proteins, amino acids, etc., as well asartificially synthesized polypeptides and a variety of known and novelsubstances such as polymers and lower molecular weight compounds.

[0031] In addition to the above method for screening substances, thepresent invention provides a kit for screening substances useful as theeffective component of prophylactic and therapeutic drugs inneurodegenerative diseases. A variety of screening kits may beconsidered; those comprising a VCP-immobilized carrier and a labeledaberrant protein are exemplified.

[0032] In such a kit, a candidate substance that is expected to inhibitthe binding of VCP and the aberrant protein is mixed with a labeledaberrant protein agent; the mixture is poured onto a VCP-immobilizedcarrier and allowed to stand for a certain period of time, the carrieris washed with a solvent, etc., and whether the candidate substance actseffectively or not maybe the presence of labeled signals on the carrieris observed to confirm confirmed by observing the presence of labeledsignals on the carrier. Generation of the labeled signal on the carrierindicates that the aberrant protein binds to VCP immobilized on acarrier, confirming that the screened candidate substance is notappropriate as a prophylactic or therapeutic drug in neurodegenerativediseases. To the contrary, no generation of the labeled signal by thecarrier indicates that the binding of VCP with the aberrant protein isinhibited, confirming that the candidate substance is appropriate as aprophylactic or therapeutic drug in neurodegenerative diseases.

[0033] In such a kit for screening, the VCP-immobilized carrier may beof any material or form on which VCP can be immobilized, including gel,film, sheet, beads, column, and the like. VCP may be immobilized on thecarrier by any means, and a variety of forms such as direct chemicalbonding, bonding via segments such as a tag, physical inclusion tobeads, etc. may be exemplified. The aberrant protein reagent may be areagent containing a protein with an extended polyglutamine chain suchas MJQ79 (causative protein of Machado-Joseph disease), or a protein forwhich abnormal conformation is confirmed. In addition it may containsolvents, buffers, additives, etc. The labeling of the aberrant proteinmay be achieved using any form of labels, for example, various tags suchas glutathione S-transferase (GST) or fluorescent proteins, orradio-isotopes. In addition to the VCP-immobilized carrier and thelabeled aberrant protein agent, the present kit for screening substancesmay contain solvents, buffers, additives, various instruments orequipment, etc. The actual process by which the binding of VCP and anaberrant protein is confirmed is not limited to the above procedures andmay include various experimental procedures such as protein synthesis,expression of fused proteins, purification by centrifugation or HPLC,electrophoresis such as SDS-PAGE, blotting, etc.

[0034] The kit for screening a substance useful as an effectivecomponent for prophylactic or therapeutic drugs for neurodegenerativediseases of the present invention may be comprised of a labeled VCPagent and an aberrant protein immobilized on a carrier. By using such akit, a substance effective as a component for prophylactic ortherapeutic drugs for neurodegenerative diseases may be screen by thesame process as in the above-exemplified kit. In other words, thelabeled VCP agent is mixed with a candidate substance that is expectedto inhibit the binding of VCP and the aberrant protein; the mixture ispoured onto an aberrant protein immobilized on a carrier and kept underappropriate conditions for a certain period of time, after which thesolution is washed out; by observing the presence or absence of thesignal from the label, the binding of VCP to the aberrant protein on thecarrier is confirmed. Hence, whether the candidate substance effectivelyacts as prophylactic or therapeutic drugs against neurodegenerativediseases as expected may be confirmed.

[0035] Detection of the labeled signal on the carrier indicates thebinding of VCP with the aberrant protein, demonstrating that thecandidate substance does not inhibit the binding and is not effective asa component for prophylactic or therapeutic drugs for neurodegenerativediseases. On the other hand, observation of no labeled signal on thecarrier indicates that the candidate substance inhibits the binding ofVCP and the aberrant protein, demonstrating that the candidate substancesufficiently works as a component for prophylactic or therapeutic drugsof neurodegenerative diseases.

[0036] The form and material of the carrier, and the components otherthan the labeled VCP in the reagent are not limited for such kit, aswell. Further, the actual procedure by which the binding of VCP and theaberrant protein is confirmed is not limited to the above-describedmeans, and various experimental procedures such as synthesis ofproteins, expression of fused proteins, purification by centrifugationor HPLC, electrophoresis such as SDS-PAGE, blotting, and the like, maybe included. Of course, the kit for screening substances of the presentinvention may contain instruments, reagents, and solvents necessary forconducting these procedures.

[0037] Modes of the present invention is described in further detailwith reference to the attached Figures. Of course, the present inventionis not limited to the following examples, and various embodiments ondetails are available.

Examples Example 1 Aggregation of Polyglutamine Due to Binding of VCPand a Protein Containing Polyglutamine Chain

[0038] PC12 cells were fixed, stained using anti-VCP antibody as thefirst antibody and FITC-labeled anti-rabbit antibody as the 2ndantibody, and observed under a fluorescent microscope to investigate thelocalization of intrinsic VCP. FIG. 1-1 shows pictures under afluorescent microscope (a) and an optical microscope (b). Thus, it wasconfirmed that VCP was expressed throughout the cells.

[0039] Next, tetracycline was eliminated from the culture medium ofPC12/Q79, and 79-repeat-polyglutamine (Q79) with Flag tag was expressed.After 48 and 96 hours from the elimination of tetracycline, the cellswere fixed, and the localization of the intrinsic VCP and polyglutaminewere observed. The cells were stained with anti-VCP antibody(FITC-labeled anti-rabbit antibody was used as the second antibody) andanti-Flag antibody (texas red-labeled anti-mouse antibody was used asthe second antibody), and the expression of VCP and polyglutamine aswell as their localization were observed.

[0040] The fluorescent micrograph are shown in FIGS. 1-2 a to c (after48 hours) and FIGS. 1-3 a to c (after 96hours). The optical micrographsof PC12/Q79 cells after expression of polyglutamine are shown in FIG.1-2 d (after 48 hours) and FIG. 1-3 d (after 96 hours).

[0041] From FIG. 1-2, it was confirmed that polyglutamine formsaggregates in the cytoplasm following induction of expression (FIG. 1-2b), and VCP co-localizes with poly-glutamine (FIG. 1-2 c).

[0042] From FIG. 1-3, it was confirmed that polyglutamine formsaggregates in the nuclei, as well, with the passage of time (FIG. 1-3b), and that VCP also co-localizes with the polyglutamine in the nuclei(FIG. 1-3 c).

[0043] Moreover, from observations under an optical microscope,vacuolization in the cytoplasm was confirmed with the expression ofpolyglutamine (FIG. 1-2 d, FIG. 1-3 d).

Example 2 Binding of VCP with Aberrant Protein MJDQ79 and Confirmationof Binding Inhibition by Chaperone Proteins (Hsp70, HJD-2)

[0044] (1) As an aberrant protein containing an extended polyglutaminechain, the causative protein of Machado-Joseph disease, MJDQ79, whichcontains 79 glutamine residues, was selected.

[0045] MJDQ79 was expressed as a fusion protein of glutathioneS-transferase (GST) in Escherichia coli.

[0046] VCP was expressed in vitro using a lysate of reticulocytes andlabeled with ³⁵S.

[0047] GST-MJDQ79 bound on glutathione agarose gel beads and ³⁵S-VCPwere mixed and slowly incubated at 4° C. (2 hours); washing andcentrifugation were repeated, and the protein that precipitated with thebeads were separated by SDS-PAGE and subjected to autoradiography.

[0048] (2) Chaperone proteins (Hsp70, HJD-2) were added to GST-MJDQ79along with VCP to confirm whether or not the binding of VCP and MJDQ79is inhibited.

[0049] The result of autoradiography is shown in FIG. 2a. The amount ofmaterials added to the reaction medium is represented based on the inputamount of the materials, which is expressed as 1. The conditions forlanes 1 to 13 are shown in Table 1. TABLE 1 Input GST GST-MJDQ79 Lane 12 3 4 5 6 7 8 9 10 11 12 13 VCP 1 0 0 5 0 0 1 1 1 0 1 1 0 Hsp70 0 1 0 05 0 0 1 5 5 0 0 0 HJD-2 0 0 1 0 0 5 0 0 0 0 1 5 5

[0050] The amount of VCP in lanes 7, 8, 9, 11 and 12 in FIG. 1a isexpressed numerically in the graph of FIG. 2b. The vertical axis isrepresented with the amount of VCP added in the input expressed as 100.

[0051] As can be seen from FIG. 2a, expressing the amount of VCP as 1,when the amount of Hsp70 was increased from 0 to 1 and 5, the VCP bandbecame weaker (FIG. 2a; lanes 7, 8 and 9). The VCP band also becameweaker as the amount of HJD-2 was increased from 0 to 1 to 5 (FIG. 2a:lanes 7, 11 and 12). The decrease in the concentration of these bandswas also confirmed from the graph in FIG. 2b. In other words, it wasconfirmed that the interaction of GST-MJDQ79 and VCP decreases with theincrease in amount of Hsp70 and HJD-2 addition.

[0052] From the above results, it was confirmed that the binding of VCPand MJDQ79 is inhibited by Hsp70 and HJD-2. Further, it was suggestedthat such inhibition was caused by the respective binding of Hsp70 andHJD-2 to GST-MJDQ79 (FIG. 2a: lanes 10 and 13).

[0053] (3) The MJDQ79 protein was labeled with ³⁵S and synthesized invitro. Further, VCP was expressed as a fusion protein with GST inEscherichia coli.

[0054] By the same process described in (1), the binding of GST-VCP and³⁵S-MJDQ79 was confirmed.

[0055] The result of autoradiography is shown in FIG. 2c. The inputamount of materials is expressed as 1, and the amount of materials addedto the reaction medium are represented based on the input. Conditionsfor lanes 1 to 14 are shown in Table 2. TABLE 2 Input GST GST-VCP Lane 12 3 4 5 6 7 8 9 10 11 12 13 14 MJDQ79 1 0 0 5 0 0 1 1 1 5 5 5 0 0 Hsp790 1 0 0 5 0 0 5 0 0 5 0 5 0 HJD-2 0 0 1 0 0 5 0 0 5 0 0 5 0 5

[0056] The amount of MJDQ79 in lanes 10, 11 and 12 of FIG. 2c isexpressed numerically in FIG. 2d. The vertical axis is represented withthe amount of MJDQ79 added in the input expressed as 100.

[0057] As can be seen from FIG. 2c, compared to the band of GST-VCP incoexistence with MJDQ79 alone, the band was weaker when Hsp7O was incoexistence (FIG. 2c: lanes 10 and 11). Additionally, the band ofMJDQ79, GST-VCP and HJD-2 also became weaker than that of MJDQ79 alonein coexistence with GST-VCP (FIG. 2c: lanes 10 and 12).

[0058] Such decrease in the concentration of the band was also confirmedfrom the graph in FIG. 2d. In other words, it was demonstrated that theinteraction of MJDQ79 and GST-VCP decreases in the system wherein Hsp70and HJD-2 were added.

[0059] From the above results, it was confirmed that the binding of VCPand MJDQ79 is inhibited by Hsp70 and by HJD-2. Further, it was suggestedthat such inhibition was caused by the respective binding of Hsp70 andHJD-2 to GST-VCP (FIG. 2c: lanes 13 and 14).

Example 3 Relaxation Mechanism of Polyglutamine InducedNeurodegeneration by Chaperone Proteins (Hsp70 and HJS-2)

[0060] It has been reported that neurodegeneration caused bypolyglutamine can be alleviated by excess expression of a chaperoneproteins such as Hsp701 or HJD-2 (Hsp40family) (Warrick, J. M., Chan, H.Y., Gray-Board, G. L., Chai, Y., Paulson, H. L., Bonini, N.M., Nat.Genet. 23, 425-428 (1999); Kazemi-Esfarijani, P., Benzer, S., Science287, 1837-1840 (2000); Muchowski, P. J., Schaffar, G., Sittler, A.,Wanker, E. E., Hayer-Hartl, M. K., Hartl, F. U., Proc. Natl. Acad. Sci.USA, 97, 7841-7846 (2000)). However, the mechanism by which suchproteins inhibit the polyglutamine neurodegeneration has not been knownup to now.

[0061] From the results of the above Example 2, it was indicated thatHsp70 and HJS-2 independently bind to VCP and thepolyglutamine-containing protein and competes with the binding of VCPand the aberrant protein to inhibit the binding in a dose-dependentmanner.

[0062] Accordingly, a mechanism wherein the chaperone protein inhibitsthe binding of VCP to polyglutamine, whereby reducing the toxicity ofpolyglutamine, was elucidated.

[0063] From the above results, changing the amount or combination ofchaperone proteins may enable stronger inhibition of the binding.

[0064] In other words, it was confirmed that the inhibition of thebinding of VCP with polyglutamine is an effective indicator in screeningdrugs that reduce the toxicity of polyglutamine.

Example 4 The Binding of VCP with a Precursor Protein of Amyloidβ-Protein

[0065] The precursor of amyloid β-protein (APP: in this experiment, thesequence from the β-secretase cleavage site to the C-terminal isindicated), which is known as the causative protein of Alzheimer'sdisease, was synthesized in vitro and labeled with ³⁵S. GST-VCP wasmixed with ³⁵S-APP, and the mixture was incubated at 4° C. for 2 hours,separated by SDS-PAGE, and subjected to autoradiograhy.

[0066] A protein containing an extended poly-glutamine (MJDQ79) was usedas a positive control, and a protein containing no domain of glutamineregion (MJDΔQ) was used as a negative control.

[0067] The results of autoradiography are shown in FIG. 3. As shown inthe figure, the lanes indicate, from left to right, the Input(synthesized protein), precipitate with GST alone, and precipitate withGST-VCP.

[0068] As in Examples 1 and 2, it was confirmed that the MJD protein iscapable of binding with VCP in the presence of polyglutamine, but not inthe absence of polyglutamine.

[0069] Similarly, it was confirmed that the aberrant protein AAP bindsto VCP.

[0070] Therefore, it was suggested that the screening of substances thatinhibit the binding of the aberrant protein (APP) and VCP is effectiveeven for the identification of substance effective as prophylactic ortherapeutic drugs for Alzheimer's disease, a neurodegenerative diseasescaused by an aberrant protein other than those containing an extendedpolyglutamine chain.

Example 5 Binding of VCP and Lewy Bodies in Dementia with Lewy Bodies(DLB)

[0071] Lewy bodies collected from a patient suffering from DLB, dementiawhere in addition to lesion in the brainstem as in Parkinson's disease,the appearance of Lewy bodies in the cerebral cortex occurr, werestained with an anti-VCP antibody

[0072] As shown in FIG. 4, the Lewy bodies were stained with anti-VCPantibody.

[0073] From this result, the presence of binding between VCP with theLewy bodies, which is a phenotype of DLB, was confirmed. Therefore, itis suggested that identification of substances useful as the effectivecomponent of prophylactic or therapeutic drugs for DLB would be possibleby screening substances that inhibit the binding.

Example 6 Binding of VCP with a Ubiquitin-Positive Nuclear InclusionBody of Spinal Cerebral Degeneration

[0074] The ubiquitin-positive nuclear inclusion body in a patient withspinal cerebral degeneration that occurred in infancy and rapidlyprogressed was stained with anti-ubiquitin antibody and anti-VCPantibody, respectively.

[0075] From FIG. 5, it was confirmed that the ubiquitin-positive nuclearinclusion body is stained with anti-ubiquitin antibody (FIG. 5a) as wellas with anti-VCP antibody (FIG. 5b).

[0076] From this result, the binding of VCP and the ubiquitin-positivenuclear inclusion body of spinal cerebral degeneration was confirmed.Therefore, it is suggested that by screening substances that inhibitsuch binding, identification of substances that are useful as theeffective component of prophylactic or therapeutic drugs against spinalcerebral degeneration would be possible.

INDUSTRIAL APPLICABILITY

[0077] As described above in detail, the present invention provides amethod for screening substances useful as the effective component ofprophylactic or therapeutic drugs for neurodegenerative diseases. Theinvention also provides a kit for screening substances effective ascomponents in prophylactic or therapeutic drugs for neurodegenerativediseases. Research, development and commercialization of prophylactic ortherapeutic drugs for neurodegenerative diseases can be expected toaccelerate, by using the screening method and the screening kit of thepresent invention.

1. A method for screening substances which are useful as effectivecomponents of prophylactic or therapeutic drug for neurodegenerativediseases caused by the binding of an aberrant protein and avalosin-containing protein, comprising: making the aberrant protein andthe valosin-containing protein and the candidate substance coexist; andidentifying the substance that shows inhibitory action on the binding ofthe aberrant protein and the valosin-containing protein.
 2. The methodfor screening a substance of claim 1, wherein the aberrant protein is aprotein contains an extended glutamine-chain.
 3. The method forscreening a substance of claim 1, wherein the aberrant protein containsan abnormal conformation.
 4. The method for screening a substance ofclaim 1, wherein the aberrant protein is a protease-resistant protein.5. The method for screening a substance of claim 1, wherein the aberrantprotein contains a P-sheet structure.
 6. The method for screening asubstance of any one of claims 1 to 5, wherein the aberrant protein andthe valosin-containing protein and the candidate substance are made tocoexist by mixing in a solution.
 7. The method for screening a substanceof any one of claims 1 to 5, wherein the aberrant protein and thevalosin-containing protein and the candidate substance are made tocoexist in a cultured cell.
 8. The method for screening a substance ofany one of claims 1 to 5, wherein the aberrant protein and thevalosin-containing protein and the candidate substance are made tocoexist by administering each component to an animal.
 9. The method forscreening a substance of any one of claims 1 to 8, wherein theinhibitory action is detected by: labeling either the aberrant proteinor the valosin-containing protein; immobilizing the non-labeled proteinon a carrier; contacting the aberrant protein and the valosin-containingprotein in the presence of the candidate substance; and detecting thesignal generated by the labeled protein on the immobilized carrier. 10.A kit for screening substances which are useful as effective componentsof prophylactic or therapeutic drug for neurodegenerative diseasescaused by the binding of an aberrant protein and a valosin-containingprotein, comprising: a carrier on which the valosin-containing proteinis immobilized; and a labeled aberrant protein reagent.
 11. A kit forscreening substances which are useful as effective components ofprophylactic or therapeutic drug for neurodegenerative diseases causedby the binding of an aberrant protein and a valosin-containing protein,comprising: a labeled valosin-containing protein reagent; and a carrieron which the aberrant protein is immobilized.